Adhesion to the extracellular matrix and the ability to move is key for survival, proliferation, and development of all higher eukaryotic cells. In turn, the assembly of focal adhesions is regulated by phosphoinositide signal transduction. In phosphoinositide signaling, phosphatidylinositol is ! phosphorylated on the three, four, or five hydroxyl of the myo-inositol ring and then by the phosphatidylinositol-phosphate kinases (PIPK), forming all known phosphatidylinositol-bisphosphates (PIP2) and phosphatidylinositol-trisphosphate (PIP3). These are essential as precursors to many . phosphatidylinositol-derived messengers and as direct messengers. Using information from the PIPK structure, we have defined the mechanism underlying the broad substrate specificity. The type I PIP kinases (PIPKI) synthesize PI4, 5P2, which is known to modulate actin and focal adhesion assembly. One isoform, the PIPKIa, has roles in PDGF-stimulated actin ruffling; another, the PIPKIy, is tightly associated with focal adhesions. Both PIPKJ isoforms are modulated by small 0-proteins. We hypothesize that these PIPKI isoforms regulate membrane ruffling and focal adhesion function; their signaling and regulation are key to understanding these processes. This hypothesis will be critically assessed with the following Specific Aims: 1. The molecular mechanisms for signaling specificity and regulation of PIPKIs by lipid mediators and G-proteins will be investigated using a structure-function approach. The substrate specificity toward PI3P and PI4P will be defined by mutagenesis of the activation loop. Regulation by lipid modulators will be examined. A focused study will map the small G-protein interacting region within PIPKIalpha. 2. The participation of PIPKIa in PDGF-induced membrane ruffle actin assembly will be investigated. Does PDGF-induced membrane ruffling require PIPKIa? The mechanisms of PIPKIcx L regulation in PDGF-stimulated actin foci assembly will be examined, with a focus on the small G-proteins. 3. The impact of PIPKIgamma660 on the assembly and function of focal adhesions will be investigated. The targeting of PIPKIgamma660 to focal adhesions will be defined, as will the role of PIPKIgamma660 in modulating the focal adhesions and cell adhesion and cell spreading on specific extracellular matrices. As movement and adhesion to the extracellular matrix clearly control cell growth and likely the invasiveness of tumor cells, these investigation have potential to impact proliferative diseases and cancer metastasis.